細(xì)胞ERK通路及其關(guān)鍵激酶MEK不同亞型在調(diào)控單純皰疹病毒Ⅱ型復(fù)制中作用研究
發(fā)布時(shí)間：2019-03-04 19:43
【摘要】：目的： 本研究通過闡明細(xì)胞Raf/MEK/ERK(簡稱ERK)通路在調(diào)控單純皰疹病毒Ⅱ型(herpes simplex virus type 2, HSV2)復(fù)制周期中的作用,并在此基礎(chǔ)上以ERK通路關(guān)鍵激酶MEK蛋白為切入點(diǎn),解析MEK1和MEK2亞型在由HSV2介導(dǎo)的ERK通路活化過程中不同激活時(shí)相中的作用,以期深入了解MEK激酶不同亞型的功能,為揭示ERK信號通路參與調(diào)控HSV2復(fù)制的作用機(jī)制提供重要的實(shí)驗(yàn)依據(jù)。 方法： 1.通過觀察病毒感染宿主細(xì)胞引起的細(xì)胞病變效(cytopathetic effect, CPE),建立HSV2感染HEK293細(xì)胞(human embryonic kidney 293 cells)的病毒—細(xì)胞模型； 2.用MOI=1.5的HSV2感染HEK293細(xì)胞,收集感染后Omin、5min、10min、20min、30min以及1h、2h、4h、8h、12h、24h、48h時(shí)間點(diǎn)的細(xì)胞及上清液,用Western blot方法檢測細(xì)胞中ERK蛋白的磷酸化水平,即p-ERK1/2(phospho-ERK1/2,p-ERK1/2)水平,用終點(diǎn)滴定法檢測上清液中病毒感染滴度(50% tissue culture infectious dose,TCID50)觀察HSV2復(fù)制對ERK通路活化的影響； 3.用MEK1/2特異性抑制劑U0126預(yù)處理HEK293細(xì)胞1h,再用MOI=1.5的HSV2感染細(xì)胞,分別收集與方法2相同的感染時(shí)間點(diǎn)的細(xì)胞及上清液,檢測各時(shí)間點(diǎn)p-ERK1/2水平及上清液中病毒TCID50,觀察U0126對ERK通路活化的影響及阻斷ERK通路對HSV2復(fù)制的影響； 4.在距離紫外燈管17cm處用紫外線照射(Ultraviolet-irradiated, UV) HSV230min將其滅活,即UV-HSV2(以下也稱為死病毒)。然后用MOI=10的死、活病毒分別感染HEK293細(xì)胞,收集感染后20min和4h的細(xì)胞,檢測各時(shí)間點(diǎn)p-ERK1/2水平,以觀察死、活病毒對ERK通路不同激活時(shí)相的影響； 5.分別設(shè)計(jì)并合成針對人類mek1、mek2基因的特異性的小干擾RNA (smallinterfering RNA, siRNA)及其突變型MKE1 siRNA (mutant MKE1 siRNA, siMut1)、MKE2 siRNA (mutant MKE2 siRNA,siMut2),用INTERFERin轉(zhuǎn)染試劑盒將其分別轉(zhuǎn)染HEK293細(xì)胞48h后,再分別用MOI=10的死、活病毒感染細(xì)胞,收集感染后20min、4h的細(xì)胞并用Western blot方法檢測p-ERK1/2水平,觀察分別敲降(knock down) MEK1、MEK2后對病毒感染引起的ERK通路早期時(shí)相和晚期時(shí)相激活的影響。 結(jié)果： 1.HSV2能夠感染HEK293細(xì)胞并引起典型的細(xì)胞病變效應(yīng),HEK293細(xì)胞可以作為易感細(xì)胞用于后續(xù)實(shí)驗(yàn)； 2.HSV2感染可引起HEK293細(xì)胞的ERK通路激活,且呈雙相激活,p-ERK1/2水平分別在病毒感染后20min和411達(dá)到高峰,可選用這兩個(gè)時(shí)間點(diǎn)分別代表ERK通路激活的早期和晚期時(shí)相； 3.U0126可以顯著抑制HSV2感染引起的ERK通路中的p-ERK1/2水平的增高,且抑制ERK通路能夠明顯抑制細(xì)胞上清液中的病毒滴度水平； 4.HSV2活病毒感染能引起ERK通路早期時(shí)相和晚期時(shí)相激活,而死病毒僅能引起ERK通路早期時(shí)相激活； 5.單獨(dú)敲降細(xì)胞MEK1對活病毒引起的ERK通路的早期時(shí)相和晚期時(shí)相激活均有顯著抑制作用,且對死病毒引起的早期時(shí)相的激活有抑制作用。而單獨(dú)敲降細(xì)胞MEK2對活病毒引起的早期時(shí)相和晚期時(shí)相激活及死病毒引發(fā)的晚期時(shí)相激活均未見顯著影響。 結(jié)論： 1.HSV2的復(fù)制依賴細(xì)胞ERK通路的激活； 2.HSV2感染宿主細(xì)胞引起ERK通路雙相激活的機(jī)制是不同的,ERK通路的早期時(shí)相激活可能與病毒的吸附、穿入和脫殼過程相關(guān)；而晚期時(shí)相激活可能與病毒在宿主細(xì)胞中的生物合成過程相關(guān)。因此,細(xì)胞ERK通路的不同激活時(shí)相可能是其調(diào)控HSV2復(fù)制一種重要機(jī)制； 3.MEK1激酶在HSV2介導(dǎo)的ERK通路雙相激活中發(fā)揮了關(guān)鍵的正調(diào)控作用,其調(diào)控機(jī)制可能是通過MEK1-ERKs級聯(lián)激活方式來調(diào)控的HSV2介導(dǎo)的ERK通路雙相激活。
[Abstract]:Purpose: In this study, the role of Raf/ MEK/ ERK (ERK) in the replication cycle of herpes simplex virus type 2 (HSV2) was clarified, and the key kinase MEK protein of ERK was cut in. The role of the MEK1 and MEK2 subtypes in the activation of the ERK pathway mediated by HSV2 in the activation of the ERK pathway mediated by the HSV2 is analyzed, with a view to further understanding the function of the different subtypes of the MEK kinase, and to provide an important experimental basis for revealing the mechanism of the ERK signaling pathway to participate in the regulation and regulation of the replication of the HSV2. It was reported. Methods:1. The virus of HEK293 cells (HEK293 cells) infected with HSV2 was established by observing the cytopathogenic effect (CPE) caused by the host cell of the virus. 2. The HEK293 cells were infected with HSV2 with MOI = 1.5, and after infection, Omin, 5min, 10min, 20min and 30min were collected. The level of ERK protein was detected by Western blot, that is, p-ERK1/2 (phospho-ERK1/2, p-ERK1/2). The HSV2 replication pair ERK is observed by the DSE (TCID50) Effect of pathway activation;3. The HEK293 cells were pre-treated with MEK1/2-specific inhibitor U0126 for 1 h, and the cells were infected with HSV2 with MOI = 1.5, and the cells and the supernatant at the same time of infection as method 2 were collected, and the p-ERK1/2 level and the supernatant at each time point were detected. The effect of U0126 on the activation of ERK pathway and the blocking of ERK via TCID50 The effect of HSV2 replication;4. Inactivation by UV-irradiation (UV) HSV230min at a distance of 17 cm from the UV lamp, i.e., UV-HSV 2 (also referred to as dead virus). Then, with MOI = 10, the live virus was infected with HEK293 cells respectively, and the cells of 20 min and 4 h after infection were collected, and p-ERK1/2 levels were detected for each time point to observe the death and live virus to ERK. The effect of the different activation phases of the path;5. The specific small interfering RNA (siRNA) and its mutant MKE1 siRNA (siMu1) and MKE2 siRNA (mutant MKE1 siRNA, siMu2) for human mek1 and mek2 genes were designed and synthesized. Cells were collected for 20 min and 4 h after infection, and the level of p-ERK1/2 was detected by Western blot. phase-time phase Results:1. HSV2 can infect HEK293 cells and cause typical cytopathic effect, HEK29 3 cells can be used as susceptible cells for subsequent experiments;2. The HSV2 infection can cause the ERK passage of the HEK293 cells to be activated and the two-phase activation, p-ERK1/2 levels, respectively, The two time points can be selected when the peak is reached at 20 min and 411 after the virus infection 3. U0126 can significantly inhibit the level of p-ERK1/2 in ERK pathway caused by HSV2 infection and inhibit ERK. the pathway can significantly inhibit the level of virus titer in the supernatant of the cell;4. the HSV2 live virus infection can cause the early phase and the late phase of the ERK pathway Phase activation, whereas the dead virus can only cause early phase activation of the ERK pathway;5. The individual knock-down cell MEK1 is responsible for the live virus The early phase and late phase activation of ERK pathway were significant. Inhibition, and inhibition of the activation of the early phase caused by the dead virus, while the individual knock-down cell MEK2 has an early phase and a late phase of the live virus. phase-time phase There was no significant effect on the activation and late phase activation of the activation and death of the virus Conclusion:1. The replication of HSV2 depends on the activation of ERK pathway. And the early phase activation of the ERK pathway may be associated with the adsorption, penetration, and dehulling of the virus. Range-related; while late-phase activation may be related to the process of the biosynthesis of the virus in the host cell. The different activation phases of the ERK pathway may be an important mechanism for regulating the replication of HSV2; K1 kinase plays a key role in the two-phase activation of the ERK pathway mediated by HSV2, and its regulatory mechanism may be through ME
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R373
本文編號：2434571
[Abstract]:Purpose: In this study, the role of Raf/ MEK/ ERK (ERK) in the replication cycle of herpes simplex virus type 2 (HSV2) was clarified, and the key kinase MEK protein of ERK was cut in. The role of the MEK1 and MEK2 subtypes in the activation of the ERK pathway mediated by HSV2 in the activation of the ERK pathway mediated by the HSV2 is analyzed, with a view to further understanding the function of the different subtypes of the MEK kinase, and to provide an important experimental basis for revealing the mechanism of the ERK signaling pathway to participate in the regulation and regulation of the replication of the HSV2. It was reported. Methods:1. The virus of HEK293 cells (HEK293 cells) infected with HSV2 was established by observing the cytopathogenic effect (CPE) caused by the host cell of the virus. 2. The HEK293 cells were infected with HSV2 with MOI = 1.5, and after infection, Omin, 5min, 10min, 20min and 30min were collected. The level of ERK protein was detected by Western blot, that is, p-ERK1/2 (phospho-ERK1/2, p-ERK1/2). The HSV2 replication pair ERK is observed by the DSE (TCID50) Effect of pathway activation;3. The HEK293 cells were pre-treated with MEK1/2-specific inhibitor U0126 for 1 h, and the cells were infected with HSV2 with MOI = 1.5, and the cells and the supernatant at the same time of infection as method 2 were collected, and the p-ERK1/2 level and the supernatant at each time point were detected. The effect of U0126 on the activation of ERK pathway and the blocking of ERK via TCID50 The effect of HSV2 replication;4. Inactivation by UV-irradiation (UV) HSV230min at a distance of 17 cm from the UV lamp, i.e., UV-HSV 2 (also referred to as dead virus). Then, with MOI = 10, the live virus was infected with HEK293 cells respectively, and the cells of 20 min and 4 h after infection were collected, and p-ERK1/2 levels were detected for each time point to observe the death and live virus to ERK. The effect of the different activation phases of the path;5. The specific small interfering RNA (siRNA) and its mutant MKE1 siRNA (siMu1) and MKE2 siRNA (mutant MKE1 siRNA, siMu2) for human mek1 and mek2 genes were designed and synthesized. Cells were collected for 20 min and 4 h after infection, and the level of p-ERK1/2 was detected by Western blot. phase-time phase Results:1. HSV2 can infect HEK293 cells and cause typical cytopathic effect, HEK29 3 cells can be used as susceptible cells for subsequent experiments;2. The HSV2 infection can cause the ERK passage of the HEK293 cells to be activated and the two-phase activation, p-ERK1/2 levels, respectively, The two time points can be selected when the peak is reached at 20 min and 411 after the virus infection 3. U0126 can significantly inhibit the level of p-ERK1/2 in ERK pathway caused by HSV2 infection and inhibit ERK. the pathway can significantly inhibit the level of virus titer in the supernatant of the cell;4. the HSV2 live virus infection can cause the early phase and the late phase of the ERK pathway Phase activation, whereas the dead virus can only cause early phase activation of the ERK pathway;5. The individual knock-down cell MEK1 is responsible for the live virus The early phase and late phase activation of ERK pathway were significant. Inhibition, and inhibition of the activation of the early phase caused by the dead virus, while the individual knock-down cell MEK2 has an early phase and a late phase of the live virus. phase-time phase There was no significant effect on the activation and late phase activation of the activation and death of the virus Conclusion:1. The replication of HSV2 depends on the activation of ERK pathway. And the early phase activation of the ERK pathway may be associated with the adsorption, penetration, and dehulling of the virus. Range-related; while late-phase activation may be related to the process of the biosynthesis of the virus in the host cell. The different activation phases of the ERK pathway may be an important mechanism for regulating the replication of HSV2; K1 kinase plays a key role in the two-phase activation of the ERK pathway mediated by HSV2, and its regulatory mechanism may be through ME
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R373
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中國期刊全文數(shù)據(jù)庫 前2條
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2 王波;丁麗新;鄧娟;張浩;朱萌;易婷;劉靜;徐萍;魯鳳民;彭宜紅;;MEK1/2抑制劑U0126抑制腸道病毒71型的復(fù)制[J];中國生物化學(xué)與分子生物學(xué)報(bào);2010年06期
，本文編號：2434571
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